Genes for biosynthesis of tetracycline compounds and uses thereof

ABSTRACT

The invention relates to tetracycline products produced by genetically engineered cells, and to therapeutic methods using such tetracyclines. The present invention is based on the cloning and heterologous expression of genes encoding the chelocardin biosynthetic pathway.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/536,622 filed Aug. 6, 2009, now U.S. Pat. No. 8,361,777, which claimsthe benefit of EP Application No. 08014141.9 filed Aug. 7, 2008, theentire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to genetically engineered cells, and to proteinsand genes useful in the production of tetracycline compounds, to methodsof producing tetracycline compounds, and to tetracyclines therebyproduced. The present invention is based on the cloning and heterologousexpression of genes encoding the chelocardin biosynthetic pathway.

BACKGROUND OF THE INVENTION

Tetracyclines are a large group of drugs with a common basic structureconsisting of four linearly fused six-membered rings. Chlortetracyclineisolated from Streptomyces aureofaciens was introduced in 1948 andoxytetracycline, derived from Streptomyces rimosus, was introduced in1950 (Projan et al., 2006. SIM News 55, 52-60). Tetracycline and6-demethyl-7-chlortetracycline (demethylchlortetracycline), bothproduced by Streptomyces aureofaciens, are two additional tetracyclinecompounds produced by fermentation process. A number of semi-synthetictetracyclines generated by chemical modification of tetracycline ordemeclocycline and with improved pharmacological properties, have beengenerated over the years such as methacycline, doxycycline andminocycline. Recently, a novel semisynthetic analogue, tigecyclin,derived from minocycline has been licensed for treatment of bacterialinfections (Chopra et al., Roberts M. 2001. Microbiol Mol Biol Rev;65:232-260). Tetracyclines were the first broad-spectrum antibiotics.They are effective against a variety of microorganisms and are thusoften used indiscriminately. Tetracyclines bind reversibly to the 30Ssubunit of the bacterial ribosome in a position that blocks the bindingof the aminoacyl-tRNA to the acceptor site on the mRNA-ribosome complex.They are bacteriostatic for many gram positive and gram negativebacteria, including some anaerobes, for rickettsiae, chlamidiae,mycoplasmas and L-forms, and for some protozoan parasites. Thewidespread use of tetracyclines has led to the emergence of resistanceeven among highly susceptible species such as pneumococci and group Astreptococci. For this reason a novel antibacterial is needed;tetracyclines, as relatively safe antibiotics, still representpotentially useful candidates for antibacterial drug discoveryprogrammes. The tetracycline analogue doxycycline has been used fordecades for the inhibition of the malaria-causing Plasmodium falciparum.Novel tetracycline (TC) analogues have been developed in the past(Projan et al., 2006. SIM News 55, 52-60). A small group of TCanalogues, of which the primary target is not a bacterial ribosome, suchas chelocardin and 6-thiatetracycline, have been isolated or synthesised(Chopra I. 2004. Antimicrob Agents Chemother; 38(4):637-40).

This small group of TC analogues are bactericidal, rather thanbacteriostatic. Their mode of action is clearly not oriented towards abacterial ribosome. It is believed that the primary target of thesesmall group of tetracycline analogues, such as chelocardin, is thebacterial cytoplasmic membrane, hence the activity of these compoundsagainst tetracycline resistant strains. This has been suggested in thestudy by Olivia et al. (1992, Antimicrob Agents Chemother. 36(5):913-919), in which the activities of these tetracycline analogues wereexamined against E. coli and Staphylococcus aureus strains containingdeterminants for efflux Tet(B) and Tet(K) or ribosomal protectionTet(M). Chopra et al. (2001, Curr Opin Pharmacol. 1(5):464-9) havedemonstrated that Tet(B) and Tet(M) determinants in E. coli orStaphylococcus aureus offer little or no protection against thetetracycline analogues chelocardin and 6-thiatetracycline, thusrepresenting an interesting antibacterial activity. Unfortunately, theclinical trials conducted with one of these atypical TCs(6-thiatetracycline) have revealed adverse side-effects. LD50 of both,6-thiatetracycline and chelocardin, obtained in acute toxicity studiesin mice, are considerably lower than those obtained by classical TCs,possibly reflecting the membrane-disruptive properties of atypical TCs.In view of this potential mode of action it is not surprising that theatypical TCs exhibit activity against TC-resistant strains. Therefore,the use of atypical TCs (such as chelocardin) can not be consideredbecause of their potential for causing side effects. Selectedtetracycline analogues have also displayed potent antifungal activity.Several chemically modified tetracycline analogues (CMTs), which werechemically modified to eliminate their antibacterial efficacy, such asCMT3, were found to have potent antifungal properties (Liu et al., 2002,Antimicrob agents chemother 46, 1447-1454).

Tetracycline analogues, including the medically important tetracyclineanalogues, show other, non-antibacterial pharmacological properties,therefore showing useful activity for the treatment of chronicneurodegenerative diseases (Parkinson's, Huntington's) and autoimmunecondition multiple sclerosis (Domercq and Matute, 2004, Trends PharmacolSci. 2004. 25(12):609-12, Brundula et al., 2002, Brain 125: 1297-308).In addition, some of the TCs inhibit the activity of matrixmetalloproteinases (MMPs), which play an important role in thedevelopment of atherosclerosis, rheumatoid arthritis, osteoporosis,tumour invasion and metastasis (cancer development/progression) (Fife etal., 2000, Cancer Lett. 29; 153(1-2):75-8). Pathologies that areresponsive to tetracycline compounds include inflammatoryprocess-associated states. The term “inflammatory process-associatedstate” includes states involving inflammation or inflammatory factorssuch as MMPs.

Some of these MMPs break down fibrillar collagens and are known ascollagenases (e.g. MMP-1, MMP-8 and MMP-13), some can affect basementmembrane collagen (collagen IV) and are known as gelatinases (MMP-2 andMMP-9). Tetracycline analogues can inhibit both collagenases andgelatinases (Peterson J. T. 2004, Heart Fail Rev., 9, 63-79).MMPs-degrading enzymes (e.g. MMP-8, MMP-9), present in the intracellularmatrix of tissue facilitate angiogenesis by allowing new blood vesselsto penetrate into the matrix. Currently only Periostat® (CollaGenexPharmaceuticals Inc.), also known as doxycycline, is approved fortreatment of adult peridontitis, as an MMP inhibitor. Theanti-angiogenic effect of tetracyclines may have therapeuticimplications in inflammatory processes accompanied by new blood vesselformation, as it is the case in some stages of autoimmune disorders andcancer invasion. Metastat (Col-3), for example, has demonstrated goodresults in the treatment of Karposi's sarcoma (Phase II, Dezube et al.,2006, J Clin Oncol. 24(9):1389-94). TCs can also influence bonemetabolism. Prophylactic administration of doxycycline reduces theseverity of canine osteoarthritis in the dog anterior cruciate model (Yuet al., 1992. Arthritis Rheum. 1992 October; 35(10):1150-9). In a recentexperiment it was demonstrated that minocycline, by stimulating new boneformation, prevents the decrease in mineral density (osteoporosis)observed in ovariestomized old rats (Wiliams et al., 1998. Adv Dent Res.1998 November; 12(2):71-5), suggesting the potential use of TCs in thetreatment of osteoporosis. Nevertheless, tetracyclines have been shownto demonstrate anti-inflammatory properties, antiviral properties andimmunosuppressive properties. The tetracycline analogue minocycline, forexample, is considered as a safe effective treatment for patients withmild to moderate rheumatoid arthritis. Tilley et al. 1995 (Ann InternMed., 122, 2. 1995, 81-89.) carried out a clinical trial in which 109patients on minocycline were compared to 110 patients on placebo. Therewas a significant improvement in joint swelling in the treated patientsversus the placebo group and also improvement in joint tenderness, withno serious toxicity.

To date, all clinically useful TC antibiotics are either naturalproducts, semisynthetic analogues, or chemically modified molecules,composed of four rings, designated A, B, C, and D (FIG. 1). The recentlyestablished crystal structure of tetracycline (TC)-bound 30s subunit(Brodersen et al., 2000, Cell, 103:1143-54.) has revealed that the sideof the four-member ring structure of TC molecule, including carbons C1to C3 and C10 to C12 (“south” and “east” side) interact significantlywith the ribosome. Most semisynthetic tetracycline analogues withsuperior antibacterial activity, such as doxycycline, minocycline andthe latest derivative tigecycline, have been modified at the“north-west” side of the tetracycline structure, covering carbons C4 toC9, which is in line with the structure-activity (SAR) results(Brodersen et al., 2000, Cell, 103:1143-54). The structure ofchelocardin, in particular, differs from existingbiosynthetically-derived natural tetracyclines, thus allowing novelchemistry to be carried out on the tetracycline backbone of chelocardinor modified matrices generated by biosynthetic-engineering approaches,which is the main scope of the invention. Combined synthetic andbiosynthetic complementary strategies for novel TC compounds can beapplied. The four ring naphtacene nucleus structure of chelocardin and acomplex series of oxygen functional groups on the “south” side of themolecule fulfil the minimal structural requirements for bioactivityagainst both bacterial and mammalian targets. However, the structure ofchelocardin is extremely non-polar, compared to other biosynthetic TCderivatives, which is a consequence of the lack of hydroxyl groups atpositions C5 and C6, and the replacement of the amino group of the amidemoiety at the position C2 with acyl. An addition, the methyl-group atthe position C9 further enhances the non-polar properties of chelocardinat the same time altering/broadening the spectrum of biological targets,not only limited to bacterial cells. On the other hand, the freeamino-group, not found “unprotected” in other natural tetracyclineanalogues introduces a degree of polarity. At the same time, it is oneof the most useful functional groups that can be readily derivatized bya chemical synthesis approach, introducing changes in solubility,lipophilicity and new binding affinities into the molecule.

In the past, extensive data on the structure-activity relationship ofTCs have been generated, showing that the molecular structure andfunctionality of different TCs allows them to be “chemicallypromiscuous” and interact with many macromolecules, hence exerting abroad spectrum of pharmacological effects. The present invention isrelated to the generation of novel TC analogues based on chelocardinitself and/or chelocardin analogues generated by methods of biosyntheticengineering, biotransformation and/or semisynthetic approaches. A moredetailed description of the present invention is provided herein below.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of chelocardin.

FIG. 2 shows the cloning of the pLUS02 cosmid used for constructing agenomic library of A. sulphurea.

FIG. 3 shows the chelocardin biosynthesis pathway, according to theinvention.

FIG. 4 shows the chelocardin biosynthetic gene cluster, and genesinvolved in chelocardin production. These are drawn as full arrows. Thethree genes on the left side of the cluster (white arrows) are involvedin the B12 biosynthesis, coding for cobalamin biosynthesis protein,cobyric acid synthase 1 and cobyric acid synthase 2 (in order from leftto right). On the right side of the chelocardin cluster genes (grayarrows), there are again several genes presumably not involved in thechelocardin biosynthesis. Restriction sites within the nucleotidesequence of the chelocardin cluster are marked as E=EcoRI, H=HindIII,B=BglII, S=SphI, N=NcoI, Sac=SacI, S3AI=Sau3AI.

FIG. 5 shows a generic tetracycline structure based on the chelocardintetracycline backbone.

FIG. 6 shows preferred groups of enzymes of the invention, together withtheir respective biosynthetic products.

SUMMARY OF THE INVENTION

The present invention relates to the application of biosyntheticengineering for the heterologous expression of an entire gene clusterfor the biosynthesis of chelocardin or its analogues, either produced byAmycolatopsis sulphurea ATCC NRRL2822 or heterologously expressed in asurrogate host. The chelocardin gene cluster is cloned and can beexpressed in a heterologous actinomycete host such as Streptomyceslividans, Streptomyces albus, Streptomyces rimosus, Amycolatopsisorientalis and Nocardia spp.

The present invention relates to processes and materials (includingprotein kits, DNA kits, nucleic acids, vectors and cells and cultures)for the heterologous expression of various Type-II polyketide geneclusters, such as the one involved in the biosynthesis of chelocardin.The present invention also relates to the preparation of novelsubstituted tetracycline compounds. The invention provides the entirenucleic acid sequence of the biosynthetic gene cluster for chelocardinproduction in Amycolatopsis sulphurea, and the use of all or part of thecloned DNA to produce novel chelocardin analogues in Amycolatopsissulphurea or surrogate hosts. A previously unknown biosynthetic pathwayfor the biosynthesis of chelocardin was identified (see FIG. 3). Newdrug candidates can be obtained by the genetic manipulation of thediscovered biosynthetic genes. Additional genes or inactivation ofselected chelocardin-pathway encoding genes is used to modify thestructure of the obtained tetracycline compound. Cells and nucleic acidsof the invention can be used for the preparation of modified chelocardinmolecules with alternative biological activities, such as antibacterial,antimalarial, antitumor, etc. agents.

Tetracycline compounds of the present invention are useful for treatmentof bacterial and fungal infections, treatment of malaria, as atherapeutic in the treatment of inflammatory process-associated statessuch as cancer, periodontitis, osteoarthritis, rheumatoid arthritis,autoimmune condition multiple sclerosis and other pharmacologicalactivities/pathologies such as cardiovascular and neurodegenerativedisorders (Alzheimer's disease & Huntington's disease). The use ofproducts produced by method of the current invention for the treatmentof any of the medical indications stated above is also covered by thepresent invention.

The present invention also relates to a treatment for inhibitingmicrobial, fungal, antiviral and tumour growth, tumour invasion andmetastasis, malaria causing protozoan parasites of the genus Plasmodium,and for a treatment of pathological conditions such as atherosclerosis,rheumatoid arthritis, multiple sclerosis, osteoporosis and usefulactivity for the treatment of chronic neurodegenerative diseases(Parkinson's, Huntington's). Chelocardin-derived matrices generated thisway are useful for generating potential compounds or intermediatessuitable for further modification by semi-synthetic or biotransformationapproach. Designer tetracycline analogues can be applied using arational approach by modifying the initiation module in the biosyntheticroute in order to replace the methyl group of the acyl moiety at theposition C2 with an amino group, thus resulting in an amide moiety. Anumber of positions on the chelocardin skeleton, such as positions C3,C4, C5, C6, C7, C8 and C9 can likely be modified using a rational or acombinatorial biosynthetic approach as well as a biotransformationapproach, in order to produce novel TC-matrices, suitable substrates forfurther chemical modifications. Using combined approaches, biosyntheticand synthetic approaches, numerous chelocardin tetracycline analogues,with potential novel activity can be generated.

By applying biosynthetic engineering approach designated positions(R1-R7 and OR; FIG. 5) can be modified by manipulation of DNA sequenceencoding chelocardin biosynthesis and its co-expression withheterologous-genes from other tetracycline and more widelyaromatic-polyketide (Type II) PKS gene clusters such as oxytetracycline,chlortetracycline, tetracenomycin, and others.

The chelocardin biosynthetic pathway according to the invention is shownin FIG. 3. The polyketide skeleton of chelocardin is assembled from anacetoacetate starter unit to which 8 malonate-derived acetate buildingblocks are attached by the action of the minimal PKS, namely ChdP, ChdK,ChdS. The polyketide chain is further subjected to methylation, C-9ketoreduction, and cyclisation/aromatisation, by the action of the ChdMImethyltransferase gene, the ChdT ketoreductase, and the ChdQIIcyclase/aromatase, respectively. After the cyclisation/aromatisation iscompleted by ChdQI and ChdX, the nascent aromatic compound is subjectedto post-PKS reactions, i.e. oxidations, C-4 amination, and C-9methylation, catalysed by three oxygenases ChdOI/ChdOII/ChdOIII,aminotransferase ChdN, and methyltransferase ChdMII, respectively.

TABLE 1  Genes of the Chelocardin Gene Cluster SEQ ID Gene Length LengthStart Stop NO: Name Gene Function Start Stop (bp) (AA) Codon Codon 1chdP Ketosynthase-alpha 85 1384 1299 433 GTG TGA 2 chdKKetosynthase-beta 1408 2635 1227 409 GTG TGA 3 chdS Acyl Carrier Protein2662 2926 264 88 ATG TGA 4 chdQI Cyclase 3879 2973 906 302 ATG TGA 5chdQII Cyclase Aromatase 17617 18562 945 315 ATG TGA 6 chdXCyclase/Aromatase 16635 16183 453 151 ATG TGA 7 chdL Acyl-CoA Ligase15753 GTG 8 chdT Ketoreductase 16800 17589 789 263 ATG TGA 9 chdOIOxygenase 11901 13113 1212 404 ATG TGA 10 chdOII Oxygenase 18530 198201290 430 ATG TGA 11 chdOIII Oxygenase 14436 14229 207 69 ATG TAG 12chdMI Methyltransferase 14149 13126 1023 341 ATG TGA 13 chdMIIMethyltransferase 4975 3970 1005 335 GTG TGA 14 chdN Aminotransferase11724 10425 1299 433 GTG TGA 15 chdGIV Glycosyltransferase 5162 63591197 399 ATG TGA 16 chdTn Transposase 6584 8099 1515 505 ATG TGA 17 chdRExporter 9686 8243 1443 481 ATG TGA 18 chdA Transcriptional Regulator9836 10406 570 190 GTG TAA

DETAILED DESCRIPTION OF THE INVENTION

“% identity”, within the context of the present invention shall beunderstood the % identity of two sequences as calculated by theNCBI/BLAST (blastx or blastp) algorithm using the default parameters asset by the NCBI (Altschul et al., 1990, J Mol Biol, 215, 403-10).

“Stringent conditions”, within the meaning of the invention, shall beunderstood as being the stringent conditions as set forth according toSambrook et al. (2^(nd) ed., 1989, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.)

A “tetracycline compound” according to the invention is a compoundhaving the chemical structure shown in FIG. 5, with R1-R7 and OR beingarbitrary substituents. Preferred tetracyclines of the inventions arethose specifically disclosed herein.

A “genetically engineered cell”, within the meaning of the invention, isa cell which is modified by purposeful application of recombinant DNAtechnology to provide said cell with modified biochemical properties.

A “genetically engineered biosynthetic pathway”, within the meaning ofthe invention, is a biosynthetic pathway which is modified by purposefulapplication of recombinant DNA technology, wherein said purposefulapplication of recombinant DNA technology involves addition of furthergenes/proteins/reactions to the pathway, and/or the deletion orinactivation of genes/proteins/reactions from the pathway, in order toobtain a modified biosynthetic pathway having improved performance or amodified biosynthetic pathway product.

A “catalytic function” of a polypeptide is understood to be the abilityof said polypeptide to catalyse a certain biochemical reaction, orbiological process.

A “gene cluster”, within the meaning of the invention, shall beunderstood to be a totality of DNA coding for polypeptides required tocatalyse a certain biochemical pathway. A gene cluster can be on asingle DNA molecule, or can be on multiple DNA molecules, e.g. in formof a DNA library.

The present invention relates to a genetically engineered cell,

-   -   said cell being capable of producing a tetracycline compound,    -   said tetracycline being produced by said cell by a genetically        engineered biosynthetic pathway,    -   wherein said genetically engineered biosynthetic pathway        includes at least one reaction catalysed by a polypeptide        selected from the group consisting of:        -   (a) a polypeptide of the chelocardin biosynthetic pathway,            said polypeptide having the sequence of any of SEQ ID NO:1            to 18;        -   (b) a polypeptide which is at least 80%, 90%, 95%, 99%,            99.9%, or 100% identical to a polypeptide of (a), wherein            said polypeptide being 80%, 90%, 95%, 99%, 99.9%, or 100%            identical has the same catalytic function as said            polypeptide of (a).

This genetically engineered cell is thus purposefully engineered toprovide a modified tetracycline biosynthetic pathway, which pathwayincludes at least one of the previously unknown genes of the chelocardinbiosynthetic pathway provided by the present invention.

Engineered cells of the invention may comprise a single one of the newlyfound genes or proteins of the chelocardin biosynthetic pathway, or theymay include multiple or all of said newly found genes or proteins of thechelocardin biosynthetic pathway (SEQ ID NO:1 to 18). The person skilledin the art appreciates that any one of the genes or proteins of thepresent invention can be substituted or replaced by homologous genes, ifthese genes show the same catalytic function. Genetically engineeredcells comprising such homologous genes or polypeptides are thus also anaspect of the invention.

The invention further relates to a genetically engineered cell asdescribed above, wherein said genetically engineered biosyntheticpathway of said cell further includes at least one reaction catalysed bya polypeptide selected from the group consisting of

-   -   (c) a polypeptide of any of SEQ ID NO:19 to 26;    -   (d) polypeptide which is at least 80%, 90%, 95%, 99%, 99.9%, or        100% identical to a polypeptide of any of SEQ ID NO:19 to 26,        wherein said polypeptide being 80%, 90%, 95%, 99%, 99.9%, or        100% identical has the same catalytic function as said        polypeptide of SEQ ID NO:19-26.

Polypeptides of SEQ ID NO:19-26 are previously known enzymes thecatalytic function of which can advantageously be applied in geneticallyengineered cells of the above described type. Notably, addition of thesepolypeptides allows to produce modified tetracycline compounds, such ase.g. the structures shown in the present application denoted Structure 1to Structure 10, below.

The present invention further relates to a genetically engineered cellof the above kind, wherein said genetically engineered biosyntheticpathway of said cell includes all reactions catalysed by a polypeptideof a group consisting of

-   -   (c) a polypeptide of any of SEQ ID NO:1 to 8;    -   (d) polypeptide which is at least 80%, 90%, 95%, 99%, 99.9%, or        100% identical to a polypeptide of any of SEQ ID NO:1 to 8,        wherein said polypeptide being 80%, 90%, 95%, 99%, 99.9%, or        100% identical has the same catalytic function as said        polypeptide of SEQ ID NO:1-8.

Polypeptides according to SEQ ID NO:1-8 represent a minimum set ofproteins capable of producing the core tetracycline compound shown asStructure 10. Genetically engineered cells of this type can also be usedas a starting point for the purposeful manipulation of such cells, toproduce modified tetracycline compounds.

The present invention also relates to a polypeptide selected from thegroup consisting of:

-   -   (a) a polypeptide of the chelocardin biosynthetic pathway, said        polypeptide having the sequence of any of SEQ ID NO:1 to 18;    -   (b) a polypeptide which is at least 80%, 90%, 95%, 99%, 99.9%,        or 100% identical to a polypeptide of the chelocardin        biosynthetic pathway having a sequence of any of SEQ ID NO:1 to        18, wherein said polypeptide being 80%, 90%, 95%, 99%, 99.9%, or        100% identical has the same catalytic function as said        polypeptide of the chelocardin biosynthetic pathway.

Such polypeptides of the chelocardin biosynthetic pathway are notpreviously not known. They can be used, individually or in combination,for the creation of new genetically engineered cells having modifiedtetracycline biosynthetic pathways, thus producing useful tetracyclinecompounds. In particular, they are useful for producing geneticallyengineered cells of the above described kind.

The present invention also relates to a nucleic acid encoding apolypeptide of the above described kind. Such nucleic acid can be inform of a single DNA molecule, or can be in form of multiple DNAmolecules, or can be in form of a gene library, or can be in form of aplasmid, or in form of multiple plasmids. Such nucleic acid can be inisolated form, or can be recombinant DNA.

The present invention also relates to an entire gene cluster encoding atetracycline biosynthetic pathway, said gene cluster comprising nucleicacid coding for

-   -   (a) polypeptides according to each one of SEQ ID NO:1 to 8, or    -   (b) polypeptides which are at least 80%, 90%, 95%, 99%, 99.9%,        or 100% identical to each one of SEQ ID NO:1 to 8, wherein said        polypeptides being 80%, 90%, 95%, 99%, 99.9%, or 100% identical        have the same catalytic function as said polypeptides of SEQ ID        NO:1 to 8.

Said gene cluster in preferably in isolated form.

The present invention further relates to a gene cluster of the abovedescribed kind, said gene cluster further comprising nucleic acid codingfor a polypeptide selected from the group consisting of:

-   -   (c) polypeptides according to SEQ ID NO:9 to 18, and    -   (d) polypeptides which are at least 80%, 90%, 95%, 99%, 99.9%,        or 100% identical to SEQ ID NO:9 to 18, wherein said polypeptide        being 80%, 90%, 95%, 99%, 99.9%, or 100% identical have the same        catalytic function as said polypeptides of SEQ ID NO:9 to 18.

The present invention further relates to a method for the biosyntheticproduction of a tetracycline compound, said method comprising the stepsof

-   -   providing a genetically engineered cell of the above described        kind,    -   providing a substrate compound,    -   incubating said substrate compound with said genetically        engineered cell under permissible conditions, and    -   obtaining said tetracycline compound.

The present invention further relates to a tetracycline compoundproduced by this method.

The present invention further relates to a tetracycline compound of theabove described kind, wherein said compound has a structure selectedfrom the group consisting of

The present invention also relates to the individual compounds ofStructure 1 to Structure 10.

It will be understood by the person skilled in the art that the exactposition of double bonds in the above structures is cannot not always beexactly defined, due to spontaneous chemical rearrangement and/orpossible tautomeric forms/isomers which can also influence the oxidativestates of hydroxyl and keto groups at carbons 10, 11, 12 and carbon 1.It is understood that reference to the above structures, within thecontext of the present application, also refers to their tautomericforms and isoforms, as explained above.

The present invention also relates to the use of these compounds for thetreatment of bacterial or fungal infections, treatment of malaria, aneurodegenerative disease, Parkinson's disease, Huntington's, disease,periodontitis, an autoimmune condition, multiple sclerosis,atherosclerosis, rheumatoid arthritis, osteoporosis, tumour invasion,cancer and inflammatory states.

The present invention further relates to the use of a geneticallyengineered cell of the above described kind for the production of amedicament for treatment of bacterial or fungal infections, treatment ofmalaria, a neurodegenerative disease, Parkinson's disease, Huntington's,disease, periodontitis, an autoimmune condition, multiple sclerosis,atherosclerosis, rheumatoid arthritis, osteoporosis, osteoarthritis,tumour invasion, cancer and inflammatory states.

Polypeptides of SEQ ID NO:9-11 are oxygenases and are used in theproduction of Structures 1-9 as set out in FIG. 6 in the presentinvention. At least one of these enzymes is preferably included in agenetically engineered cell of the invention.

Polypeptides of SEQ ID NO:12-15 are also present in the wild typechelocardin biosynthetic gene cluster, thus, they are naturallyoptimized to co-operate with proteins of SEQ ID NO:1-11. At least one ofthese enzymes is preferably included in a genetically engineered cell ofthe invention.

Polypeptides of SEQ ID NO:16-18 are enzymes not involved in thebiosynthetic pathway, but supporting the functioning of the enzymes ofthe SEQ ID NO:1-8 in a cellular environment (e.g. by transportingmetabolites across cell membranes, by providing resistance to the finalproduct, by activation of protein expression, and by other favourableeffects). At least one of these enzymes is preferably included in agenetically engineered cell of the invention.

Polypeptides of SEQ ID NO:19-26 are heterologous enzymes not comprisedin the wild type chelocardin biosynthetic gene cluster. They can be useto design and construct further tetracycline structures with improvedproperties, as exemplified in FIG. 6 of the present application. Atleast one of these enzymes is preferably included in a geneticallyengineered cell of the invention.

The present invention also relates to the (previously unknown) proteinsaccording to SEQ ID NO:1-18 as such, and to DNA molecules encoding thesame.

Methods of the invention are preferably conducted in a bioreactor or ina fermenter.

In the accompanying sequence listing,

-   -   SEQ ID NO:1 codes for ChdP,    -   SEQ ID NO:2 codes for ChdK,    -   SEQ ID NO:3 codes for ChdS,    -   SEQ ID NO:4 codes for ChdQI,    -   SEQ ID NO:5 codes for ChdQII,    -   SEQ ID NO:6 codes for ChdX,    -   SEQ ID NO:7 codes for ChdL,    -   SEQ ID NO:8 codes for ChdT,    -   SEQ ID NO:9 codes for ChdOI,    -   SEQ ID NO:10 codes for ChdOII,    -   SEQ ID NO:11 codes for ChdOIII,    -   SEQ ID NO:12 codes for ChdMI,    -   SEQ ID NO:13 codes for ChdMII,    -   SEQ ID NO:14 codes for ChdN,    -   SEQ ID NO:15 codes for ChdGIV,    -   SEQ ID NO:16 codes for ChdTn,    -   SEQ ID NO:17 codes for ChdR,    -   SEQ ID NO:18 codes for ChdA,    -   SEQ ID NO:19 codes for OxyD,    -   SEQ ID NO:20 codes for OxyT,    -   SEQ ID NO:21 codes for OxyE,    -   SEQ ID NO:22 codes for OxyL,    -   SEQ ID NO:23 codes for OxyS,    -   SEQ ID NO:24 codes for OxyG    -   SEQ ID NO:25 codes for Cts4, and SEQ ID NO:26 codes for TcmO.

Envisaged are further tetracycline compounds of FIG. 5 having thefollowing substituents:

-   R1: CH₃ or NH₂ (otc homologue oxyD (Zhang et. al., 2006, Appl.    Environ. Microbiol., 72(4):2573-2580); Zhang et al., 2007, J Biol.    Chem. 282(35):25727-25)-   R2: NH2 or OH. or N(CH3)₂ (inactivation of chdN or co-expression    with oxyT (Zhang et. al., 2006, Appl. Environ. Microbiol.,    72(4):2573-2580); Zhang et al., 2007, J Biol. Chem.    282(35):25727-25)-   R3: H or OH (otc co-expression with oxyE/oxL/oxyS/oxyG (Zhang et.    al., 2006, Appl. Environ. Microbiol., 72(4):2573-2580); Zhang et    al., 2007, J Biol. Chem. 282(35):25727-25)-   R4: CH3 or H or/and OH (inactivation of chdMI or co-expression with    oxyxE/oxyL/oxyS/(otcC)/oxyG (Peric-Concha et al., 2005, J. Biol.    Chem.; 280(45):37455-60; Zhang et al., 2007, J Biol. Chem.    282(35):25727-25).-   R5: H or Cl (ctc co-expression with chl (cts4) (Dairi et al, 1995,    Biosci. Biotechnol. Biochem. 59(6):1099-106))-   R6: H-   R7: CH3 or H (inactivation of chdMII)-   OR: OH or OCH3

EXAMPLES Example 1 Cloning of the Chelocardin Gene Cluster

The cloning the chelocardin cluster starts with isolating a PCR templateDNA of a chelocardin producer, Amycolatopsis sulphurea (NRRL2822),followed by a PCR using degenerate primers based on universallyconserved motifs of Type-II acyl-ketosynthase alpha (KSα) (Metsa-Ketelaet al., 1999, FEMS Microbiol Lett.; 180(1):1-6). The partial KSαnucleotide sequence of the PCR product was confirmed by sequencing. ThePCR product was used as a probe against BamHI, SacI, BglII, SphI, EcoRI,and NcoI total restriction digests of A. sulphurea genomic DNA. An 8 kbEcoRI-fragment that gave a positive result after Southern hybridizationwith a DIG-labelled KSα PCR probe was the most suitable for generating ashotgun library in pUC19. A shotgun library of app. 8 kb EcoRI digestsof A. sulphurea genomic DNA was created in pUC19. On the basis of colonyhybridization with the KSα probe, the 8 kb E1-E2 insert (FIG. 4) wasselected for sequencing.

The E1-E2 insert of the pLUC10E (FIG. 4) contains the genes clearlycoding for a Type-II polyketide cluster, namely: KSα, KSβ, ACP,cyclase/aromatase, and methyltransferase. Within that same insert, thegenes involved in B 12 vitamin biosynthesis were also found, namelycobyric acid synthase 1, cobyric acid synthase 2, and the cobalyminbiosynthesis enzyme (FIG. 4).

Anticipating that the Type-II polyketide chelocardin cluster is lessthan 30 kb in size, a cosmid library was constructed in E. coli XL1-BlueMR from genomic DNA derived from a chelocardin producer, Amycolatopsissulphurea (NRRL 2822). The cosmid library was screened by colonyhybridization using a single KSα probe obtained by PCR using degeneratedoligonucleotides, generated on the basis of conserved KSα nucleotidesequences (Metsa-Ketela et al., 1999, FEMS Microbiol Lett.; 180(1):1-6).

An integrative conjugative cosmid vector pLUS02 (FIG. 2) was firstconstructed as a tool to create a cosmid library and to speed up thesubsequent conjugation and heterologous expression of the many positiveclones, found by colony hybridization using a single probe.

The pLUS02 cosmid was used as the basis for the Amycoltopsis sulphureagenomic library. For constructing the pLUS02 (FIG. 2), the commercialSuperCos1 vector (Stratagene) that contains an E. coli origin ofreplication, a selectable drug resistance marker, and the cos sites ofphage lambda was used. SuperCos1 itself cannot be transferred between E.coli and Streptomyces, and is unable to replicate in Streptomyces. Tocircumvent this deficit, SuperCos1 was upgraded with the insertion ofthe “oriT, attP, int” cassette. The oriT of an IncP transmissibleplasmid promotes the transfer of DNA by conjugation from an E. colidonor strain to an Streptomyces/Actinomyces host recipient. The attP andthe integrase from actinophage ΦC31 enable the site-specific integrationof the conjugated DNA into the phage attachment site in a Streptomyceschromosome (Bierman et al., 1992, Gene 116:43-49). Therefore, noadditional subcloning is needed for heterologous expression afteridentifying the relevant cosmids within the cosmid library.

By upgrading the commercial SuperCos1 vector with the “oriT, attP, int”cassette, we created a vector pLUS02 that enables us to carry out fasterand easier functional analyses of multiple cosmids. The cloning capacityof the 11.6 kb pLUS02 cosmid vector is 33.4 kb, which, together withother properties, renders this tool suitable for cloning andheterologous expression of the many Type-II polyketide clusters.

The pLUS02-based genomic library of Amycolatopsis sulphurea was screenedfor the presence of the chelocardin gene cluster using a Type-II PKSprobe (KSα). Eighteen positive clones were selected out of 1600 coloniesthat were hybridized. At this point, additional PCR screening wasperformed using the primers based on the pLUC10E, selecting the clonesthat did not have a cobyric acid synthase 2 (left side of thechelocardin gene cluster, FIG. 4), while at the same time did contain amethyltransferase (ChdMII, FIG. 4). On the basis of the PCR screen andend-sequencing of the cosmids, the VII C4 cosmid was selected,sequenced, and roles of the gene products were determined in silico.

The genes involved in the biosynthesis of chelocardin have beenheterologously expressed in different Streptomyces and non-streptomycetehosts. For the production, vegetative and production media, togetherwith the method for extraction, were optimized for the strains.

An HPLC method was carried out to collect fractions of the extracts ofthe chosen host productive strains carrying the entire gene clusterencoding for chelocardin biosynthesis. Biological activities of thefractions were tested against Micrococcus luteus. The active fractionswere subjected to further MS/MS analysis which confirmed the presence ofa sodium adduct of chelocardin with a corresponding mass of m/z=413 anda proton adduct m/z=412.

Example 2 Isolation of Amycolatopsis Sulphurea Genomic DNA

A 10% innoculum of Amycolatopsis sulphurea NRRL 2822 strain was grown in25 mL of Tryptone Soy Broth (Oxoid) at 30° C. for 24 h. The mycelium waswashed two times in 25 ml of TES buffer (25 mM Tris-HCl, pH8; 50 mMEDTA, pH8; 0.3 M Sucrosis), resuspended in 5 mL of TES buffersupplemented with 4 mg/mL Lyzozyme (Sigma) and 100 μg/mL RNase (Sigma),and incubated at 37° C. for one hour. Then, 170 μA of 10% SDS was added,followed by the addition of 800 μL of 0.5 M EDTA (pH8) and 20 μL ofProteinase K (Sigma). After a 30 minute incubation at 37° C., 500 μL of10% SDS was added. After one hour of incubation at 37° C., 3 mL of TEbuffer (10 mM Tris-HCl, pH8; 1 mM EDTA, pH8), 2 mL of 5 M NaCl and 5 mLof TNE buffer (10 mM Tris-HCl, pH8; 100 mM NaCl, 1 mM EDTA, pH8) wereadded, followed by phenol/chloroform (1:1) extractions and ethanolprecipitation. The DNA was eluted in 1 mL of TE buffer. The sufficientpurity and size of DNA were assayed by gel electrophoresis.

Example 3 Generating a Probe to be Used in the Search for ChelocardinGene Cluster

A homologous hybridization probe for screening A. sulphurea genomic DNAfor chelocardin was generated by PCR using degenerated oligonucleotideprimers. The primers were designed to amplify a fragment of Type-IIketosynthase alpha (KSα) gene (Metsa-Ketela et al., 1999, FEMS MicrobiolLett.; 180(1):1-6), namely: PKSF: 5′-TSGCSTGCTTCGAYGCSATC-3′ (SEQ IDNO:27), and PKSR: 5′-TGGAANCCGCCGAABCCGCT-3′ (SEQ ID NO:28), where S=Cor G; Y=C or T; B=C, G or T and N=A, T, C or G.

The PCR conditions for the amplification of KS genes were as follows.About 10 ng of purified DNA template, 1 pmol of each primer, 0.5 mMdNTP, 10% DMSO, 1× Taq polymerase reaction buffer, 2 mM MgCl2, 1 μL TaqDNA Polymerase were used in the final reaction volume of 50 μL. The PCRreaction started with a longer denaturation phase (5′/95° C.) beforeadding Taq Polymerase. Thirty cycles were set as follows: denaturation(1′/95° C.), annealing (1′/64° C.) and extension (1.5′/72° C.). Thereaction was ended with a longer final extension (10′/72° C.). Thesequence analysis of the PCR products confirmed the KSα sequence. Theproducts were DIG-labelled according to the kit manufacturer (Roche) andused as a probe for Southern hybridization.

Example 4 Southern Blots and DNA Hybridization

The DIG-labelled KSα PCR product was used for hybridization against theBamHI, SacI, BglII, SphI, EcoRI, and NcoI total restriction digests ofA. sulphurea genomic DNA, separated by gel electrophoresis (0.8% gel,for 20 h at 25V). The DNA was transferred to the positively chargedHybond-N+ membrane (Amersham Pharmacia) according to the manufacturer.

Prehybridization and hybridization were performed at 46° C., then washedtwo times for 15 minutes in 2×SSC, 0.1% SDS at room temperature and twotimes for 5 minutes in 0.2×SSC, 0.1% SDS at 45° C. The approximately 8kb EcoRI fragment proved to be the most suitable for creating a shotgunlibrary.

Example 5 Generating a Shotgun Library

A high quality A. sulphurea genomic DNA was completely digested with therestriction endonuclease EcoRI and size fractionated by gelelectrophoresis (1% agarose gel, 1.5 V/cm, 20 h). The separated DNA wasexcised from the gel in several layers, covering 6-10 kb fragments. Eachlayer of DNA was extracted from the gel using Wizard SV Gel and PCRClean-Up System (Promega). A fraction of each isolate was run on a freshgel (1% agarose, 5 V/cm, 2 h), transferred to a Hybond-N+ membrane(Amersham Pharmacia) and hybridized again using the same KSα probe toselect for the samples with the highest concentration of the desired 8kb fragment with the chelocardin cluster. The selected sample was usedin a cloning reaction with a dephosphorylated pUC19/EcoRI. The ligationmix was transformed into electrocompetent E. coli DH10β and the shotgunlibrary was searched for the presence of genes from the chelocardincluster by colony hybridization.

For colony hybridization, cells from single colonies aftertransformation were inoculated on a positively charged Hybond-N+membrane (Amersham Pharmacia), placed on top of a layer of 2TY agar(Tryptone 16 g; Yeast extract 10 g; NaCl5 g pH 7.0), supplemented withampicillin (100 mg/L). After an overnight incubation at 37° C., themembrane with grown colonies was put sequentially on Whatman papers,soaked in

-   -   10% SDS for 5 minutes,    -   0.5 M NaOH; 1.5 M NaCl for 10 minutes    -   1.5 M NaCl; 0.5 M Tris-HCl (pH7.5) for 10 minutes    -   2×SSC for 10 minutes.

Then the membrane was washed in 2×SSC containing 200 μg/mL Proteinase Kfor approximately one hour. Colony debris was removed with a glovedfinger. The membrane was briefly washed in 2×SSC, air-dried, and bakedat 80° C. for 1-2 hours. Prehybridization and hybridization wereperformed at 42° C., then washed two times for 15 minutes in 2×SSC, 0.1%SDS and two times for 15 minutes in 0.2×SSC, 0.1% SDS, both at 68° C.

Six positive clones obtained from colony hybridization of an 8 kbEcoRI-EcoRI shotgun library were subjected to additional PCR analysis,confirming the presence of KSα, a Southern blot/DNA hybridization usinga KSα probe, and end-sequencing of the inserts.

One of the positive plasmids, pLUC10E, was chosen for sequencing byprimer walking. The 8 kb EcoRI1-EcoRI2 insert revealed five open readingframes characteristic of the expected Type-II chelocardin gene cluster,namely: KSα, KSb, ACP, cyclase/aromatase, and methyltransferase. Asshown in FIG. 3A, the insert also contained 3 genes involved in vitaminB12 biosynthesis, namely cobyric acid synthase, the cobalyminbiosynthesis enzyme, and the cobalbumin biosythesis enzyme. Thischaracteristic of the insert shows that this is one end of the cluster,which was exploited as described later.

Example 6 Construction of a Conjugable Integrative Cosmid

The 3.7 kb PvuII-BamHI fragment containing the “oriT, attP, int”cassette from pQ803 (A. Almuteirie, PhD Thesis, 2006, University ofStrathclyde) was blunt-ended by Klenow and subcloned intoSmaI-linearized and dephosphorylated SuperCos1 (FIG. 2). The efficiencyof conjugation of the two cosmids with different orientations of the“oriT, attP, int” cassette, namely pLUS01 and pLUS02, was compared. Theconjugation efficiency was expressed as the ratio of exconjugants (S.coelicolor M145) per donor (E. coli ET12567/pUZ8002), both determined bythe number of CFU. Repeatedly, the pLUS02 version of the cosmidconjugated at a higher frequency than the pLUS01 version. The pLUS02cosmid yielded 1.5*10⁻⁶ exconjugants per recipient while the pLUS01version conjugated at 7.2*10⁻⁶ exconjugants per recipient. Due to thissmall but consistent difference in conjugation efficiency, the pLUS02cosmid was selected as tool for creating a genomic library ofAmycolatopsis sulphurea.

Example 7 Cloning Packaging and Transduction

DNA isolation was carried out as described earlier (Example 1). A.sulphurea genomic DNA was partially digested with the restrictionendonuclease Sau3AI. Optimal digestion conditions to generate large DNAfragments of app. 35 kb size range were empirically determined byconducting a series of digestions, followed by the appropriate scale-up.The DNA was size fractionated by gel electrophoresis, and cloned intothe unique BamHI cloning site of a dephosphorylated pLUS02. The in vitropackaging of the ligation mix and the transduction of phage particlesinto the E. coli host XL1-B1ueMR was done according to the Gigapack IIIGold packaging kit supplier (Stratagene). The cosmid pLUS02 was used asa basic tool for the preparation of an Amycolatopsis sp. genomiclibrary. The 11.6 kb pLUS02 cosmid vector contains the lambda cos siteto promote packaging of vectors containing approximately 38 to 52 kb DNAfragments in total into phage particles. From the pLUS02-basedAmycolatopsis sp. genomic library, 60 cosmids were isolated by analkaline lysis procedure (Birnboim HC and Doly J. 1979. DNA NucleicAcids Res. 7:1513-1523). All the cosmids (60 out of 60) within the bankcontained an insert of sufficient size to fulfil the phage packagingcapacity requirements. The 60 isolated cosmids were digested with EcoRI,the restriction fragments were separated by gel electrophoresis, and thesizes of the cosmids were determined by the Quantity One Gel Docdocumentation system. All 60 isolated cosmids showed a highlyrepresentative different restriction pattern with an average size of44.9 kb, which renders the cloning capacity of the vector 33.4 kb inaverage.

Example 8 Screening for the Chelocardin Cluster by Colony Hybridization

Within the library of 2400 clones, 1600 were screened by colonyhybridization, as described previously. Cosmid DNA from approximately1600 clones was spotted onto a Hybond-N+ membrane (Amersham Pharmacia)and hybridized with random-primed DIG-labelled strain-specific Type IIPKS probe (KSα) by the following standard hybridization procedure(Roche). The membranes were prehybridized and hybridized as described.

A subset of 18 positive cosmids that were obtained on the basis ofhybridization to the KSα probe was additionally screened via three PCRs.Degenerated KSα primers (Metsa-Ketela et al., 1999, FEMS MicrobiolLett.; 180(1):1-6), methyltransferase (from the chdMII gene, FIG. 4)primers (C-MT1F: 5′-CTGCAGCCACGGCTACTAC-3′ [SEQ ID NO:29], C-MT1R:5′-GCTCGTAGGTCTTGGTCGAG-3′ [SEQ ID NO:30]), and cobalbumin biosynthesisprotein (FIG. 4) primers (CobdoF: 5′-GTGGGCCGACTCGAAGAG-3′ [SEQ IDNO:31], CobdoR: 5′-GGTTGACCAGATCGTCGGTA-3′ [SEQ ID NO:32]) were used inthe following 50 μL reaction: 1×Pfu Polymerase buffer, 0.2 mM dNTPs, 4μM forward- and reverse-primers, 5 μL od DMSO, 1 U of Pfu Polymerase, 1μL of pLUC10E DNA. The PCR thermal cycles were set as follows: aninitial cycle at 95° C. for 2 min, and 30 cycles at 95° C. for 30 s,59.2° C.-61.5° C. for 30 s, and 72° C. for 2 min, followed by a finalextension-cycle at 72° C. for 10 min. The strategy behind the PCR screenwas to choose the cosmids that give the KSα and methyltransferaseamplification products, but not the cobyric acid synthase product.Southern hybridization and end-sequencing of the selected inserts wasalso performed, and the VII C4 cosmid was chosen for sequencing.

Example 9 Identification of Gene Function in the Chelocardin GeneCluster

The open reading frames (ORFs) were analysed with the FramePlot programand gene functions were assigned according to the homology searches inthe protein database (BLASTp), supported with the conserved domainsearches.

The isolated nucleic acid comprises the genes of the chelocardin genecluster. As depicted in FIG. 4, the cluster contains 18 genes typical ofa Type-II polyketide cluster. Consistently with the chelocardinstructure, the cluster is comprised of three genes forming a “so-called”minimal Type-II PKS (KSα, KSβ, ACP), three genes involved in thecyclisation/aromatisation process, two genes for methyltransferases, onegene for aminotransferase, three genes for oxygenases, a gene for aketoreductase, a gene for an acyl-CoA ligase, a gene for a drugresistance transporter and a transcriptional regulator, as well as aglycosyltransferase and a transposase which are redundant. A briefdescription of the genes involved in the biosynthesis of chelocardinfollows:

ChdP-acyl-ketosynthase alpha

BLASTp sequence analysis shows that the chdP sequence corresponds to aketosynthase gene characteristic of Type-II PKS clusters. The tcsDketo-acylsynthase gene from the S. auerofaciens chlortetracycline genecluster showed the highest similarity (77% identity) to the chdP gene.The oxyA gene from the oxytetracycline producer S. rimosus (76%identity), and the spiramycin producer S. spiramyceticus (71% identity)followed in homology. Together with these strains, many other (mostlyType-II) polyketide producers showed more than 70% identities, such asS. echinatus (aranciamycin), S. albofaciens, S. platensis, S. fradiae(urdamycin), S. tendae (cervimycin), S. nogalater (nogalamycin). ThecmmP gene, coding for a ketosynthase in S. griseus subsp. griseus showsa 69% identity and is, according to Menendez et al. (2004, Chem. Biol.11(1):21-32) together with cmmK and cmmS, coding for a minimal PKS,responsible for the 20-C backbone of the glycosylated Type-II polyketidechromomycin. With the e-values ranging down to 4e-142, all BLASTpresults strongly suggest that chdP is coding for a keto-acyl synthasealpha (KSα). The N terminal catalytic domain of the ChdP proteinharbours a well conserved aa region around the highly conserved activesite Cys¹⁷³ (GPVGLVSTGCTSGVDVIGHA [SEQ ID NO:33]) responsible forcatalyzing the iterative condensation of the ketoacyl:ACP intermediates.In the C terminus of the protein there is an amino-acid sequencecharacteristic of the acyltransferase site (VPVSSIKSMVGHSLGAIGSLEVAA[SEQ ID NO:34]) with the active Ser³⁵¹ residue that binds to an acylchain (Fernandez-Moreno et al., 11992. J Biol. Chem. 267(27):19278-90).

ChdK-acyl-ketosynthase beta

According to BLASTp results, chdK is a keto-acyl synthase beta (KSβ),also named a chain length factor. Similarly as for KSα, S. rimosus, S.aureofaciens, S. chartreusis, S. echinatus, S. antibioticus, S.argillaceus, and S. griseus subsp. griseus return as hits with 73 to 63%aa identity. Ketosynthase domain active-site cysteine residue in ChdK isreplaced by a highly conserved glutamine as in KSQ (VSEQ¹⁸¹AGGLD [SEQ IDNO:35]) and in other chain-length factors of type II PKS synthases.According to Bisang et al. (1999, Nature 401, 502-505), the glutamineresidue is important both for decarboxylase activity and for polyketidesynthesis.

ChdS-acyl-carrier protein

The ChdS has the highest sequence similarity (59% identity) to S.rimosus acyl carrier protein (ACP), and harboures an active Ser⁴¹residue in the highly conserved motif (LGYDSL [SEQ ID NO:36]), to whichphosphopantetheine binds in order to connect the incoming extender unit(Walsh et al., 1997. Curr Opin Chem. Biol. 1(3):309-15).

ChdQI

The deduced chdQI product is involved in cyclisation/aromatization ofthe polyketide chain. The ChdQI amino acid sequence is most similar tothe CmmQI protein from S. griseus subsp. griseus, with 33% identity. ThecmmQI product codes for a cyclase/aromatase that would participate inthe cyclisation and aromatization of the first ring. It also showssimilarity to the S. argillaceus mithramycin aromatase/cyclase (mtmQI),presumably involved in C-7/C12 first ring closure. It also showssimilarity to the otcDI (also named oxyK by Zhang et al., 2006, J Nat.Prod. 69(11):1633-6; Zhang et al., 2007, J Biol. Chem. 282(35):25717-25)gene from S. rimosus. The otcDI product was identified as a bifunctionalcyclase/aromatase (Petkovie et al., 1999. J Biol. Chem.274(46):32829-34). and was proved not only to catalyze the correctformation of the D ring, but to also influence the final length of thenascent polyketide chain. A disruption of the otcDI gene in theoxytetracycline cluster leads to four truncated (by up to 10 carbons)shunt products. Within the ChdQI there are the highly conserved aminoacids, which are according to the homologous cyclise/aromatase TcmN,responsible for the determination of the final length of the polyketideand for its proper regiospecific cyclisation and aromatization (Ames etal., 2008, PNAS; 105(14): 5349-5354). These amino acids are at positionsTrp-32, Phe-36, Trp-69, Ser-71, Arg-73, Phe-92, Met-95 in Trp-99.

ChdQII

Interestingly, while ChdQI and ChdQII both align with OtcD1(orOxyK)—with 33% and 58% identity, respectively-, there is only 29%identity between the two of them. Interestingly as well is the fact thatonly chdQII shows a typical didomain architecture with N- and C-terminalhalves having a reasonable similarity to each other. ChdQII is abifunctional cyclase/aromatase that lies next to the ChdT ketoreductase,and is presumably the cyclase that is involved in D ring aromatisation.It has been previously suggested by Petkovié et al. (1999. J Biol. Chem.274(46):32829-34) that there is a mandatory functional relationshipbetween the OtcD1 cyclase/aromatase and the C-9 ketoreductase, sincedespite the lack of OtcD1, aromatic rings can still be synthesised.Similarly as in the case of ChdQI, there are the highly conserved aminoacids at positions Trp-32, Phe-36, Trp-69, Ser-71, Arg-73, Phe-92,Met-95 in Trp-99. These are, according to the homologouscyclise/aromatase TcmN, responsible for the determination of the finallength of the polyketide and for its proper regiospecific cyclisationand aromatization, either of the ring B or C (Ames et al., 2008, PNAS;105(14): 5349-5354).

ChdX

The predicted protein is homologous to Oxyl and MtmX, both presumablyinvolved in the formation of the final ring A in the biosynthesis ofoxytetracycline and mitramycin, respectively (Lombo et al., 1996, Gene.172(1):87-91; Zhang et al., 2006, J Nat. Prod. 69(11):1633-6).

ChdL

The deduced protein product of the chdL gene shows a profound amino acidsequence similarity (53% identity) to an acyl CoA ligase. Similarly toall the ORFs described (or identified) so far, this one also shows ahigh degree of similarity to one of the genes from the S. griseus subsp.griseus chromomycin and the S. argillaceus mithramycin gene clusters(Lombo et al., 1996. Gene. 172(1):87-91).

ChdMII

In accordance with the chemical structure of chelocardin, there are twomethyltransferases within the chelocardin cluster, and both of theirfunctions can be clearly resolved from the translated amino acid BLASTalignment.

The translated amino acid sequence of chdMII is most similar to theC-methyltransferases CmmMII and MtmMII of S. griseus subsp. griseus(chromomycin) and S. argillaceus (mithramycin), respectively (Lozano etal., 2000. J Biol. Chem. 275(5):3065-74; Rodriguez et al., 2004, J Biol.Chem. February 279(9):8149-58; Abdelfattah and Rohr, 2006. Angew ChemInt Ed Engl. 45(34):5685-9). The two methyltransferases methylate thearomatic C-9. Furthermore, there is no BLASTp identification of any ofthe genes for oxytetracycline since it does not contain a methyl groupat C-9. From these analyses, there is a strong indication that chdMIIcodes for a methyltransferase that methylates the aromatic C-9 ofchelocardin. Similarly as for ChdMI, ChdMII also shows a typicalglycine-rich SAM-dependent methyltransferase motif (Y/DXGXGXG [SEQ IDNO:37]) that interacts with the SAM cofactor, which is used as a sourcefor the methyl group (Martin and McMillan, 2002, Curr. Opin. Struct.Biol.; 12(6): 783-93).

ChdMI

The second methyltransferase within the chelocardin cluster is the C-6methyltransferase. As expected, ChdL BLAST similarity search returns theOxyF methyltransferase from S. rimosus (with 65% identity) whichmethylates the C-6 of pretetramid in a reaction that yields6-methyl-pretetramid (Zhang et al., 2006. J Nat. Prod. 69(11):1633-6).Moreover, this time there are no hits among any of the chromomycin ormithramycin cluster genes, since they do not have a methyl group at theC-6 position. Therefore, we presume that chdMI codes for C-6methyltransferase.

ChdN

There is only one amino-group in the chemical structure of chelocardinand chdN is the only ORF that resembles an amidotransferase, accordingto the aa sequence analysis. It is most similar (52% identity) to theamidotransferase NocG from Nocardia uniformis subsp. tsuyamanensis, aproducer of the monocyclic beta-lactam antibiotic nocardicin A. Thesecond closest hit, with 51% identity, is an aspartate/tyrosine/aromaticaminotransferase from S. fungicidicus (enduracidin), followed byaminotransferases from S. coelicolor (A3) 2 and many other differentActinomycetes species, such as Frankia sp. CcI3, Salinispora tropica,Amycolatopsis balhimycina, Nonomuraea sp. ATCC39727, etc. Although allthese amidotransferases belong to a variety of different actinomycetes,they all share more than 49% identity with ChdN. Therefore, the genechdN is an amidotransferase that catalyses the single amidation ofchelocardin at the C-4.

ChdT

The protein product of chdT is most similar to oxyJ (S. rimosus),followed by simA6 from the simocyclinone gene cluster (S. antibioticusTue6040) and aknA (S. galilaeus), all genes coding for ketoreductases.Moreover, the first 60 BLASTp hits return ketoreductases with more than40% of identity to ChdT, so there is an aromatic PKS ketoreductaseinvolved in the region-specific reduction of the C-9 carbonyl group ofthe nascent polyketide chain (Hopwood, 1997. Chem. Rev. 97:2465-2497).Two conserved domains can be found within the amino acid sequence ofChdT, namely GXGXXA (SEQ ID NO:38) and G-3X-G-X-G-3X-A-6X-G (SEQ IDNO:39), proposed to act as a NADPH-cofactor binding sites (Hopwood andSherman, 1990, Annual Review of Genetics; 24:37-66; Rawlings and Cronan,1992, J Biol. Chem.; 267(9): 5751-4).

ChdOI

The protein product of chdOI is similar to several monooxygenases, suchas oxyE, cmmOI, and mtmOI. They all exhibit significant similarity toflavin adenine dinucleotide (FAD)-dependent monooxygenases involved inthe hydroxylation of different polyketides. For OxyE it has beenpresumed by Zhang et al (1997. Chem. Rev. 97:2465-249). that thisFAD-dependent monooxygenase is involved in catalyzing the C-5 oxidationof a 5a,11a-dehydrotetracycline to yield 5a,11a-dehydrooxytetracycline.But since there is a very high degree of similarity (66% identity)between ChdOI and OxyE, and no C-5 oxidation in the chelocardin (ormithramycin or chromomycin), there is a mistake in the currentprediction for the function of the OxyE product. According toAbdelfattah and Rohr (2006. Angew Chem Int Ed Engl. 45(34):5685-9),MtmOI is responsible for hydroxylating the C4 of the premithramycinonethat eventually becomes the O-atom in the 1′-position of mithramycin.According to the conserved domain search the oxygenase belongs to thefamily of FAD-dependent monooxygenases, since at the N-terminal end ofthe protein there is a typical conserved sequence G-X-G-2X-G-3X-A-6X-G(SEQ ID NO:40, where X is any amino acid) involved in the FAD-cofactorbinding (Mason and Cammack, 1992, Annu. Rev. Microbiol.; 46:277-305).

ChdOII

The product of chdOII is most similar to OxyL, followed by an oxygenasefrom S. rochei, MtmOII from S. argillaceus and CmmOII from S. griseussubsp. griseus oxygenases.

The inactivation of mtmOII generated a non-producing mutant strain whichgenerated an unstable compound (Prado et al., 1999. Mol Gen Genet.261(2):216-25). However, Abdelfattach and Rohr (2006. Angew Chem Int EdEngl. 45(34):5685-9) provided a vague, indirect proof that the productof MtmOII is responsible for the epoxidation reaction eithersimultaneously with or shortly after the correct fourth cyclization togive the tetracyclic premithramycin framework, hence hydroxylating theC-12a of the polyketide. In the mithramycin biosynthesis, MtmOII is alsoinvolved in controlling the chain length of the growing polyketide aswell as the correct region-specificity in the cyclisation step of thefourth ring. ChdOII is according to the conserved domain search also theoxygenase from the family of FAD-dependent monooxygenases, since at theN-terminal end of the protein there is a typical conserved sequenceG-X-G-2X-G-3X-A-6X-G (SEQ ID NO:41, where X is any aminoacid) involvedin the FAD-cofactor binding (Mason and Cammack, 1992, Annu. Rev.Microbiol.; 46:277-305).

ChdOIII

ChdOIII is a very short protein that shows similarity to oxygenase OxyG(S. rimosus), a small (11-kDa) quinine-forming oxygenase, possiblyinvolved in the quinine formation of ring A in 4-keto-ATC. According tothe conserved domain search, the ChdOIII is an ABM (AntibioticBiosynthesis Monooxygenase) that does not contain an FAD binding site,neither are there any other prostetic groups, metal ions or cofactorsneeded for the molecular oxygen activaton.

ChdA

The product of the chdA gene is according to BLASTp most similar to atranscriptional regulator, namely the tetracycline repressor from theTetR family of proteins that are involved in the transcriptional controlof multidrug efflux pumps, pathways for the biosynthesis of antibiotics,differentiation processes, etc. Since proteins of the TetR family havebeen found in 115 genera of gram-positive, alpha-, β-, andgamma-proteobacteria, cyanobacteria, and archaea (Ramos et al., 2005) itis not suprising that the BLAST hits return species from several genera,including the Burkholderia, Salmonella, Klebsiella, Escherichia,Aeromonas, etc.

ChdR

The ChdR protein codes for a multidrug efflux resistance protein fromthe EmrB/QacA subfamily. With 39% identity and 58% similarity it is mostsimilar to the EmrB/QacA of Frankia alni, followed by Nocardia farcinica(with 35% identity, 55% similarity), and Salinispora arenicola (36%identity, 58% similarity). Since chelocardin's mechanism of action issupposedly different from the classical binding of tetracyclines to the30S ribosomal subunit (Chopra I. 2004. Antimicrob Agents Chemother;38(4):637-40), the mere presence of an efflux protein without theadditional ribosomal protection mechanism should suffice the resistancerequirements of the strain.

Within the chelocardin cluster (FIG. 4), two redundant genes are alsopresent. The genes chdGIV and chdTn, coding for a glycosyltransferaseand a transposase, respectively, could be an evolutionary remain of thehorizontally transferred DNA fragment from a gene cluster of aglycosylated polyketide, such as chromomycin or mithramycin. The chdGIVgene product is most similar to the CmmGIV and MtmGIVglycosyltransferases of the chromomycin (Streptomyces griseus subsp.griseus) and mithramycin (Streptomyces argillaceus) producers. It istherefore also possible that A. sulphurea has a potential to produceglycosylated chelocardin analogues. However, to our knowledge, this hasnot yet been confirmed in the literature.

Example 10 Heterologous Expression

Heterologous Hosts

A method for the heterologous expression of chelocardin in a substituteActinomyces host is provided. The cloned genes for the biosynthesis ofchelocardin have been expressed in heterologous hosts such as S.lividans TK24, S. rimosus 15883S, S. albus G148, and S. coelicolorA3(2), Amycolatopsis orientalis and Nocardia sp.

Culturing Conditions

Culture conditions were optimized for the expression of the chelocardinbiosynthetic gene cluster. The following vegetative medium was used forinoculum preparation: 3 g soy flour, 0.2 g yeast extract, 1.5 mL glucose(50%), 1 g NaCl, 0.2 g CaCO3, tap water up to 200 mL. The inoculum wasgrown for 24 hours at 30° C. on a rotary shaker at 220 rpm. Thefollowing medium was used for production: 40 g soy flour, 10 g yeastextract, 4 g CaCO3, 1 g citric acid, 5 mL glucose (50%), tap water up to1800 mL. The pH of the production medium was corrected to 7.0. Forfermentation, 50 mL of production medium was inoculated with 5% inoculumin a 500 mL Erlenmayer flask and grown at 30° C. on a rotary shaker at220 rpm for 7 days.

Extracting the Produced Chelocardin

50 mL of fermentation broth was acidified to pH 1-2 with HCl, saturatedwith NaCl, followed by two extractions with 25 mL of butanol. Theextracts were vacuum-dried and subsequently resuspended in methanol tothe desired concentration.

HPLC Analysis of the Product A reverse gradient HPLC method was devisedthat makes use of two standard mobile phases:

Mobile phase A (pH 2.75): 100 mL HPLC-grade acetonitrile, 10 mL of 1 Mammonium acetate solution and 1 mL of TFA were mixed in a total volumeof 1 L, using HPLC-grade water.

Mobile phase B (pH 2.7): 100 mL HPLC-grade water, 10 mL of 1 M ammoniumacetate and 1 mL of TFA were mixed in a total volume of 1 L, usingHPLC-grade acetonitrile.

HPLC analysis was run on a 150 mm×4.6 mm Thermo-Hypersil C18-BDS reversephase column with a silica particle size of 3 μm that has an in lineshort guard cartridge containing the same silica as the column but witha silica particle size of 5 μm. The generic HPLC gradient details areshown below:

Flow rate: 1 ml/min

Column oven temperature: 30° C.

UV wave-length analysis: 279 nm

Gradient conditions: T=0 min, 10% B; T=1 min, 10% B; T=25 min, 95% B;T=29 min, 95% B; T=29.5 min, 10% B; T=36 min, 10% B.

Bioassay on the Model Organism Microccocus luteus and Escherichia coli

The 0.5 mL HPLC fractions were collected every 30 seconds. Qualitativeagar diffusion bioassay of the fractions was performed on petri dishescontaining Micrococcus luteus and Escherichia coli as test strains.Sterile disks were impregnated with 20 μL of the collected fractions andincubated on the seeded 2TY agar for 24 hours at 37° C. The fractionsthat showed zones of inhibition were subjected to further NMR/MSanalysis.

MS Analysis of the Product

An HPLC method was carried out to collect fractions of the extracts fromcultures of heterologous expression hosts carrying the entire genecluster encoding for chelocardin biosynthesis. Biological activities ofthe fractions were tested against Micrococcus luteus. The activefractions were subjected to further LC-MS/MS analysis which confirmedthe presence of a molecule of m/z=412 that corresponds to a chelocardinmolecule with a proton adduct. For the LC-MS/MS analyses, the Agilent1100 series coupled with Watters Micromass Quattro micro detector wereused together with the Gemini 3μ C18 110A (150 ×2.1 mm (Phenomenex, USA)column were used under the following running conditions:

Column temperature: 45° C.; Mobile phase A=0.05% trifluoroaceticacid—MilliQ water, B =acetonitrile; Flow: 0.24 ml/min; Gradient:

time (min) A (%) B (%) 0 80 20 13 10 90 15 10 90 15.1 80 20 20 80 20

Injection volume: 10 μL of sample

Detector conditions:

-   -   Ionisation: ESI+    -   Method: SIR

Dwell time: 0.1 s; Cone 20 V; Capillary 3.0 kV; Extractor: 3 V; Sourcetemperature: 120° C.; Desolvation temperature: 350° C.; Cone gas flow:30 L/h; Desolvation gas flow: 500 L/h; Multiplier: 650 V.

Example 11 Generation of C9-Demethyl Chelocardin Analogue

An in-frame chdMII gene inactivation experiment is performed,eliminating any possible polar effects on chdQI. Two PCR products(chdMIIa and chdMIIb) flanking the 402 nt (134 aa) sequence of chdMIIare amplified using the primers (with restriction sites underlined):

chdMIIa-forward: (SEQ ID NO: 42, EcoRI restriction site)5′-GAATTCCCACCGTCCACATAGGAAAG-3′, chdMIIa-reverse:(SEQ ID NO: 43, ZraI restriction site) 5′-GACGTCGTGATGATCACCAATGTGCTGC,chdMIIb-forward: (SEQ ID NO: 44, SfoI restriction site)5′-GGCGCCCAGCTTCAACGACGGC-3′, chdMIIb-reverse:(SEQ ID NO: 45, EcoRI restriction site) 5′-GAATTCCGACCTCAGCGTCCACATC-3′.

The 1802 and 1282 bp products of chdMIIa and chdMIIb, respectively, areseparately cloned into the SmaI linearized and dephosphorylated pUC19and confirmed by sequencing. The EcoRI-ZraI chdMIIa and SfoI-EcoRIchdMIIb products are cloned simultaneously into the EcoRI linearized anddephosphorylated pIJ4026, creating the pIJ4026-chdMII402. The properorientation of the both inserts is assured by the one-side blunt endsproduced by the ZraI and SfoI restriction enzymes. The unaltered readingframe is confirmed after sequencing using the Vector NTO software. Theplasmid pU4026-chdMII402 is inserted into the A. sulphurea chromosome bya single cross-over homologous recombination, and transformants wereselected using erythromycin (50 μg/mL). Colonies that underwent a secondcrossover, hence shortening the wild type chdMII gene by 402 nt (134aa), are chosen on the basis of the erythromycin-sensitive phenotype.The mutants with the truncated chdMII are confirmed by southernhybridisation using the chdMII gene as a probe. The chromosomal DNAs ofthe wild type A. sulphurea and the ΔchdMII mutants are digested withEcoRI. The ΔchdMII strains show a single hybridization signal at theexpected 8,696 kbp in comparison with the two bands (of 7,580 and 1.518kbp) of the wild type. Fermentation of the mutant strains, productextraction and the MS analysis are performed as described in the Example10. The resulting molecule is a chelocardin with R7=H.

-   1) Position: R1: NH2 (otc homologue of OxyD (Zhang et. al., 2006,    Appl. Environ. Microbiol., 72(4):2573-2580)

In this case, we would express oxyD gene from oxytetracycline genecluster together with entire gene cluster encoding chelocardinbiosynthesis.

Example 12 Generation of Amide-Derived Chelocardin Analogs

Structure 1

To generate the amide-derived chelocardin analogue at the position R1,the intact oxyD gene (GenBank nucleotide sequence DQ143963, nucleotidesfrom 3686 to 5524; Zhang et al., 2006, Appl. Environm. Microbiol.;72(4):2573-2580) that is involved in the in malonamyl-CoA starter unitbiosynthesis of the OTC was cloned into the phiC31/phiBT1/pSAM-basedintegrative vector and/or replicative vector under theact/erm/erm*promoter. After the transformation of the vectors,fermentation of the productive strains, product extraction and the MSanalysis were performed as described above. The following structure isobtained:

Structure 2

Position: R2: —OH (inactivation of chdN (Zhang et. al., 2006, Appl.Environ. Microbiol., 72(4):2573-2580))

The inactivation of the chdN gene encoding C4-aminotransferase fromchelocardin cluster should, according to the prior art, result in thepresence of an OH-group at position C4.

Structure 3

Position: R2: N(CH3)₂ (co-expression with OxyT (Zhang et. al., 2006,Appl. Environ. Microbiol., 72(4):2573-2580)). In this case, we wouldexpress oxyT methyltransferase gene from oxytetracycline gene clustertogether with entire gene cluster encoding chelocardin biosynthesis.

Structure 4

Position: R3: OH (otc co-expression OxyE and/or OxyL and/or OxyS and/orOxyG (Zhang et. al., 2006, Appl. Environ. Microbiol., 72(4):2573-2580),and Theriault et al., 1982, J. Antibiot March; 35(3):364-6.) In thiscase, we would express oxyE (or oxyL, or oxyS or oxyG) hydroxylase genefrom oxytetracycline gene cluster together with entire gene clusterencoding chelocardin biosynthesis.

Structure 5

Position: R4: CH3 and OH (co-expression with OxyS (OtcC) (Peric-Conchaet al., 2005, J. Biol. Chem.; 280(45):37455-60). and Theriault et al.,1982, J. Antibiot March; 35(3):364-6) or OxyE, or OxyL, or oxyG (Zhanget al., 2006, Appl. Environ. Microbiol., 72(4):2573-2580). In this case,we would express oxyS (otcC) or oxyE, or oxyL, or oxyG hydroxylase genefrom oxytetracycline gene cluster together with entire gene clusterencoding chelocardin biosynthesis.

The stereochemistry of NH₂ in the opposite epimeric form has beendescribed by (Theriault, 1982, J. Antibiot.; 35(3):364-6). The epimericform of Structure 5 is previously unknown.

Structure 6

Position R4: H (inactivation of chdMI). (Zhang et. al., 2006, Appl.Environ. Microbiol., 72(4):2573-2580) The inactivation of the chdMI geneencoding C6 methyltransferase from chelocardine cluster should,according to the available literature and our own results, result in theabsence of a CH3-group at position C6.

Structure 7

Position R5: Cl (co-expression with chl (cts4) (Dairi et al, 1995,Biosci. Biotechnol. Biochem. 59(6):1099-106))

In this case, we would express chl gene catalysing chlorination at theposition C7 from chlortetracycline gene cluster together with entiregene cluster encoding chelocardin biosynthesis.

Structure 8

Position R7: H (inactivation of chdMII)

The inactivation of the chdMII gene encoding C9 methyltransferase fromchelocardin cluster should, according to the available literature dataand our own results, result in the absence of a CH3-group at positionC6.

Structure 9

Position OR: OCH3 (co-expression with tcmO (Summers et al., 1992, J.Bac.; 174:(6):1810-1820)

In this case, we would express tcmO gene from tetracenomycin genecluster together with entire gene cluster encoding chelocardinbiosynthesis.

The position of double bonds which would be altered after proposedmodifications at carbons C4, C5 and C6 could not be defined precisely atthis point due to possible spontaneous chemical rearrangement and/orpossible tautomeric forms/isomers which can also influence the oxidativestates of hydroxyl and keto groups at carbons 10, 11, 12 and carbon 1.

Structure 10

Using the minimum set of enzymes of the invention, the followingtetracycline structure is obtained:

Example 13 Identification of Homologues

Using bioinformatic tools, the following homologues of genes of theinvention were identified. Homologues shown in Table 3 can substitutethe original genes/sequences in methods of the invention. The “%identity” is also given, as calculated with NCBI/BLAST algorithms(blastp and blastx). The BLAST search was performed against anon-redundant protein sequences database with the default general andscoring parameters (Altschul et al., 1990, J Mol Biol, 215, 403-10).

TABLE 2 gene homologue strain % identity chdP OxyA S. rimosus 77 chdKOxyB S. rimosus 73 chdS OxyC S. rimosus 59 chdQI cyclase - like proteinStreptomyces sp. WP 33 4669 chdMII C9 - methyltransferase S. griseussubsp. griseus 44 chdN aminotransferase Frankia sp 51 chdOI OxyE -oxygenase S. rimosus 64 chdMI OxyF - C6 S. rimosus 65 methyltransferasechdOIII OxyG - oxygenase S. rimosus 58 chdL acyl-CoA ligase S. griseussubsp. griseus 54 chdX OxyI - cyclase S. rimosus 63 chdT OxyJ -ketoreductase S. rimosus 74 chdQII OxyK - cyclase S. rimosus 58 chdOIIOxyL - oxygenase S. rimosus 56

The invention claimed is:
 1. A compound having the structure

wherein R₁ NH₂; R₂ is NH₂ or OH or N(CH₃)₂; R₃ is H or OH; R₄ is CH₃ or H or OH; R₅ is H or Cl; R₆ is H; R₇ is CH₃ or H; and OR is OH or OCH₃.
 2. A tetracycline compound of claim 1, wherein said compound has structure 1 